ABSTRACT CFTR is a member of the ABC Transporter superfamily, but is the only member known to bear ion channel activity. Using a molecular evolution analysis, we have identified residues in CFTR that appear to be critical to the evolutionary transition from transporter to channel. The long-term objective is to understand how the pore of the CFTR channel changes its structure between the open and closed states, how steps in the ATP-dependent gating cycle control pore gating, and how CFTR evolved the capability to interrupt a transporter mechanism by locking the permeation pathway open, thus gaining ion channel function. This proposal will test the hypothesis that chloride channel activity evolved in CFTR by converting the conformational changes in the membrane domain associated with binding and hydrolysis of ATP at the cytoplasmic domains, as are found in true ABC Transporters, into the formation of a stable open state, by means of inter- and intra-domain interactions. Results from the previous funding period show that disruption of intradomain interactions in CFTR, at sites that are also identified as divergent between CFTR and a related ABC Transporter, dramatically alter channel behavior in terms of conductance, selectivity, pharmacology, and transitions between multiple conducting states. This renewal application proposes to use this evolutionary analysis, coupled to structure/function experiments based upon quantitative electrophysiological assays of CFTR channels expressed in oocytes, and followed by simulations of a recently published CFTR homology model, to test the importance of specific residues in CFTR in the switch from transporter to channel. Aim #1 is to identify residues that underlie the evolution of channel activity in CFTR. At sites that exhibit evolutionary divergence from transporters, the impact of mutations on channel activity will be determined. At sites predicted to interact to stabilize the open state, the rate and state-dependence of crosslinking with bifunctional sulfhydryl-modifying (SH) reagents of known length will be measured. Aim #2 is to identify residues that change conformation during channel opening. Preliminary results from normal modes analysis of a CFTR homology model suggest that at least some of the helices that contribute to the pore (#1, #3, and #6) may move during channel gating. At positions along these helices, the rate of interaction of SH reagents and Cd2+ with single engineered cysteines will be measured in the closed and open states. At sites in adjacent helices that are predicted to interact, in the homology model, the rate of disulfide trapping of double cysteines by small bifunctional SH reagents also will be measured in the closed and open states. Molecular dynamics simulations will be used to test our interpretations of movement in the pore. These studies will allow us to associate molecular motions in CFTR's pore domain with steps in the ATP-dependent gating cycle. They also will allow us to identify residues that are critical to ion channel function in CFTR, and may identify sites where drugs can be docked to lock open CFTR channels, leading to increased chloride secretion.